Zinc tolerance in populations ofDeschampsia cespitosa (Gramineae) beneath electricity pylons

Patterns of zinc tolerance were examined in eightDeschampsia cespitosa (L.)Beauv. populations from normal and zinc-contaminated soils, using solution culture methods. Zinc-tolerant populations have evolved beneath pylons, and their tolerance patterns (degree, variance, heritability) vary. Tolerance is genetically based in all of them. In contrast to the cases of other species previously reported, some within-population differences are considerable, and may be due to both heterogeneity of the soils zinc contents, various gene combinations and gene recombinations between genotypes. One control population contains many tolerant plants, an original result which is discussed.     

Sporophytic-gametophytic herbicide tolerance in sugarbeet

Summary In vitro selection procedures for herbicide tolerance were initially developed in the sporophytic generation of sugarbeet (Beta vulgaris L.), and then tested in the gametophytic generation. The primary objective of our study was to develop and evaluate in vitro techniques for identifying genotypes within heterogeneous seedling populations tolerant to specific herbicides, and to use meristematic cloning procedures to synthesize clones genetically tolerant to the herbicide. Seed from cloned selections tolerant to the herbicide ethofumesate were obtained and compared to plants from seed of the original population (using germination, central bud development, and root dry weight). Verification for in vitro selection accuracy was accomplished by pollen germination studies in the gametophyte. The results indicate that in vitro selection of germinated seedlings in the presence of the proper concentration of challenging agent can be effective in identifying genotypes tolerant to ethofumesate. Such identification was accomplished in fully differentiated tissue, but without the necessity of mature plants. Gametophytic studies, via pollen germination, indicated an association between genes operating in the sprophyte and those in the gametophyte. Cloning the seedlings identified as tolerant genotypes, and subsequent intercrossing of these clones provided a convenient method of synthesizing populations with gene frequencies shifted in the direction desired.Joint contribution of the Agricultural Research Service, USDA, and the Colorado State University Experiment Station. Published with the approval of the Director of the Colorado State University Experiment Station as Scientific Series Paper No. 2952     

Selective adaptation to copper of populations ofAgrostis tenuis andFestuca rubra (Poaceae)

Populations ofAgrostis Tenuis andFestuca rubra, tolerant and non-tolerant towards Zn and Pb, were studied in regard to their adaptability by natural selection, towards a third metal (Cu) and the time required for the appearance of such tolerance. It was found that Zn and Pb tolerant and non-tolerant populations are likely to select the character of Cu-tolerance within the course of only one generation. More specifically, Zn and Pb tolerant populations show a better adaptability compared with non-tolerant ones. The amount of tolerance to copper obtained by artificial selection is comparable with that of populations growing naturally on copper mines.     

Selection for tolerance to copper during pollen formation in Mimulus guttatus Fischer ex DC

In Mimulus guttatus, copper tolerance is determined largely by a single gene and is expressed in both the sporophyte and microgametophyte. This study explores the extent to which selection during pollen formation affects copper tolerance in the sporophytic generation. Two sets of plants heterozygous for copper tolerance, produced by reciprocal crosses between different copper-tolerant or sensitive families, and the plant on which the original observations were based, were cloned and grown in control or copper-supplemented solutions. Pollen viability and the number of tolerant progeny produced in backcrosses to sensitive plants were compared. In addition, the effect of copper treatment on pollen viability in vitro was compared for plants tolerant, sensitive and heterozygous for copper tolerance. The extent to which in vitro pollen viability decreased in response to copper treatment corresponded to the copper tolerance of the pollen source. When grown with added copper, four of the five plants showed significant reductions in pollen viability, ranging from 18% to 48% of control values. The reductions in pollen viability were correlated with an increase in tolerant progeny (r= 0.679, p=0.004). Increases in tolerant progeny could be large, ranging from 119% to 170% of that of controls, but were usually smaller than was predicted from the reductions in viable pollen. In addition, plants derived from reciprocal crosses differed significantly in the extent to which pollen viability was decreased and sporophytic tolerance increased. Thus, while selection during pollen formation could increase sporophytic tolerance, sporophytic factors, perhaps including cytoplasmic or epigenetic ones, moderated the effectiveness of pollen selection for copper tolerance.     

A field study of pollen-mediated gene flow from Mediterranean GM rice to conventional rice and the red rice weed

The objective of this study was to assess the frequency of pollen-mediated gene flow from a transgenic rice line, harbouring the gusA and the bar genes encoding respectively, -glucuronidase and phosphinothricin acetyl transferase as markers, to the red rice weed and conventional rice in the Spanish japonica cultivar Senia. A circular field trial design was set up to investigate the influence of the wind on the frequency of pollination of red rice and conventional rice recipient plants with the transgenic pollen. Frequencies of gene flow based on detection of herbicide resistant, GUS positive seedlings among seed progenies of recipient plants averaged over all wind directions were 0.036 ± 0.006% and 0.086 ± 0.007 for red rice and conventional rice, respectively. However, for both red rice and conventional rice, a clear asymmetric distribution was observed with pollination frequency favoured in plants placed under the local prevailing winds. Southern analyses confirmed the hemizygous status and the origin of the transgenes in progenies of surviving, GUS positive plants. Gene flow detected in conventional rice planted at 1, 2, 5 and 10 m distance revealed a clear decrease with increasing distance which was less dramatic under the prevailing wind direction. Consequences of these findings for containment of gene flow from transgenic rice crops to the red rice weed are discussed. The precise determination of the local wind conditions at flowering time and pollination day time appear to be of primary importance for setting up suitable isolation distances.     

Do metal-rich plants deter herbivores? A field test of the defence hypothesis

Some plant species growing on metalliferous soils are able to accumulate heavy metals in their shoots up to very high concentrations, but the selective advantage of this behaviour is still unknown. The most popular hypothesis, that metals protect plants against herbivores, has been tested several times in laboratory conditions, with contradictory results. We carried out the first large-scale test of the defence hypothesis in eight natural populations of the model Zn hyperaccumulator Thlaspi caerulescens J. and C. Presl (Brassicaceae). In two climatic regions (temperate, Belgium–Luxembourg, and Mediterranean, southern France), we worked in metalliferous and in normal, uncontaminated environments, with plants spanning a wide range of Zn concentrations. We also examined the importance of glucosinolates (main secondary metabolites of Brassicaceae) as antiherbivore defences. When exposed to natural herbivore populations, T. caerulescens suffered lower herbivory pressures in metal-enriched soils than in normal soils, both in Belgium–Luxembourg and in southern France. The trapping of gastropods shows an overall lower population density in metalliferous compared to normal environments, which suggests that herbivory pressure from gastropods is lower on metalliferous soils. In addition, foliar concentration of glucosinolates was constitutively lower in all populations from metal-enriched soils, suggesting that these have evolved towards lower investment in organic defences in response to lower herbivory pressure. The Zn concentration of plants had a protective role only for Belgian metallicolous plants when transplanted in normal soils of Luxembourg. These results do not support the hypothesis that Zn plays a key role in the protection of T. caerulescens against enemies. In contrast, glucosinolates appear to be directly involved in the defence of this hyperaccumulator against herbivores.     

Phytochelatin is not a primary factor in determining copper tolerance

Phytochelatin (PC) is involved in the detoxification of harmful, non-essential heavy metals and the homeostasis of essential heavy metals in plants. Its synthesis can be induced by either cadmium (Cd) or copper (Cu), and can form stable complexes with either element. This might suggest that PC has an important role in determining plant tolerance to both. However, this is not clearly apparent, as evidenced by a PC-deficient and Cd-sensitiveArabidopsis mutant (cad1-3) that shows no significant increase in its sensitivity to copper. Therefore, we investigated whether the mechanism for Cu tolerance differed from that for Cd by analyzing copper sensitivity in Cd-tolerant transgenics and Cd-sensitive mutants ofArabidopsis. Cadmium-tolerant transgenic plants that over-expressedA. thaliana phytochelatin synthase 1 (AtPCS1) were not tolerant of copper stress, thereby supporting the hypothesis that PC is not primarily involved in this tolerance mechanism. We also investigated Cu tolerance incad2-1, a Cd-sensitive and glutathione (GSH)-deficientArabidopsis mutant. Paradoxically,cad2-1 was more resistant to copper stress than were wild-type plants. This was likely due to the high level of cysteine present in that mutant. However, when the growth medium was supplemented with cysteine, the wild types also exhibited copper tolerance. Moreover,Saccharomyces cerevisiae that expressedAtPCS1 showed tolerance to Cd but hypersensitivity to Cu. All these results indicate that PC is not a major factor in determining copper tolerance in plants.     

Pollen-mediated transgene flow in the wind-pollinated grass species tall fescue (Festuca arundinacea Schreb.)

Information regarding gene flow in wind-pollinated, outcrossing forage grasses is essential for any future releases of value-added transgenic cultivars. Experiments on pollen dispersal was carried out by growing transgenic tall fescue (Festuca arundinacea) in a central plot, surrounded by exclosures containing recipient plants up to a distance of 200 m from the central source plants in eight directions. The central transgenic tall fescue plants carried a chimeric hygromycin phosphotransferase gene (hph) and a chimeric -glucuronidase gene (gusA). Seeds were collected from the recipient plants and germinated seedlings were used for high throughput DNA isolation and polymerase chain reaction (PCR) analysis. More than 21,000 seedlings were PCR analyzed for the experiments conducted in three years. Transgenes were detected in recipient plants at up to 150 m from the central transgenic plot. The highest transgene frequencies, 5% at 50 m, 4.12% at 100 m and 0.96% at 150 m, were observed north of the central plot, the prevailing wind direction. Lower transgene frequencies were detected in other directions, particularly at 100 m and 150 m distances. No transgene was detected at 200 m distance in any direction. Transgene flow was less effective or ineffective when recipient plants were further away from the central donor plants. Southern blot hybridization analysis confirmed the transgenic nature of the PCR positive plants. A supplementary experiment demonstrated that transgene flow can be controlled by placing transgenic plantings downwind and long distances from non-transgenic seed increases, thus allowing tall fescue breeding and transgene development programs to be conducted concurrently at the same research station.     

Cadmium and copper uptake and accumulation by Sesbania rostrata seedling, a N-fixing annual plant: implications for the mechanism of heavy metal tolerance

Sesbania rostrata, an annual tropical legume, has been found to be tolerant to heavy metals, with an unknown mechanism. It is a promising candidate species for revegetation at mine tailings. In this study, sequential extractions with five buffers and strong acids were used to extract various chemical forms of cadmium and copper in S. rostrata, with or without Cd or Cu treatments, so that the mechanisms of tolerance and detoxification could be inferred. Both metals had low transition rates from roots to the aboveground of S. rostrata. The transition ratio of Cd (4.00%) was higher than that of Cu (1.46%). The proportion of NaCl extracted Cd (mostly in protein-binding forms) increased drastically in Cd treated plants from being undetectable in untreated plants. This suggests that Cd induced biochemical processes producing protein-like phytochelatins that served as a major mechanism for the high Cd tolerance of S. rostrata. The case for Cu was quite different, indicating that the mechanism for metal tolerance in S. rostrata is metal-specific. The proportion of water-insoluble Cu (e.g. oxalate and phosphate) in roots increased significantly with Cu treatment, which partially explains the tolerance of S. rostrata to Cu. However, how S. rostrata copes with the high biotic activity of inorganic salts of Cu, which increased in all parts of the plant under Cu stress, is a question for future studies. Sesbania rostrata is among the very few N-fixing plants tolerant to heavy metals. This study provides evidence for the detoxification mechanism of metals in Sesbania rostrata.     

Copper resistance and genetic diversity inLychnis alpina (Caryophyllaceae) populations on mining sites

Copper mine populations ofLychnis alpina are shown to be significantly more resistant to increased copper concentrations compared to populations on normal soils. Data obtained from isozyme polymorphism analysis revealed that although the copper populations display considerable variation, they have lower genetic variability than the populations from normal soils, both on a local and a global scale, thus indicating a slight founder effect. Copper ecotypes inL. alpina have originated independently. The results are similar to what recently have been reported in heavy metal tolerant populations ofArmeria maritima.     

Reproductive strategy of an invasive thistle: effects of adults on seedling survival

Invasive species are known for their ability to form monocultures that exclude native species, yet intraspecific interactions among invasives have not been well studied. Cynara cardunculus (L.) is an invasive perennial thistle that establishes high-density populations in coastal California grasslands. We examined the natural distribution of C. cardunculus seedlings in an established population and found that nearly 100% of seedlings grew within 2 m of adults despite an expected distribution peak at 3 m from source plants based on measured dispersal distances. We then investigated the role of mature plants in seedling survival and establishment with regard to live vegetation, litter, and seedling distance by planting seedlings at increasing distances around adults and applying removal treatments to the focal adult rosettes. We applied control (no removal), adult rosette removal (live leaves), litter removal (dead leaves), and adult rosette plus litter removal (all aboveground plant material) treatments. Seedlings experienced a higher rate of survival, measured by senescence date, and establishment, measured by return rate the following year, with all adult rosette removal treatments. Inhibition by adult rosettes was reduced with distance to 60–80 cm from the rosette, and there was little effect of adult plants between 80 and 200 cm. These results suggest that adult rosettes may inhibit conspecific seedlings at very close distances but provide a favorable environment for seedlings within nearby interspaces. This pattern may contribute to the creation and maintenance of high-density populations in C. cardunculus. Land managers seeking to control this species may improve long-term effectiveness by expanding management efforts to include a 2 m radius around adult plants and treating within 5 months of seedling emergence to prevent recruitment rather than treating adults alone.     

Polymorphism and physiology of arsenate tolerance in Holcus lanatus L. from an uncontaminated site

The polymorphism of arsenate tolerance in a Holcus lanatus L. population from an uncontaminated soil was investigated and a high percentage of tolerant individuals (65%) was found in the population studied. Influx of arsenate was highly correlated to arsenate tolerance within the population, with the most tolerant individuals having the lowest rates of arsenate influx. Isotherms for the high affinity arsenate uptake systems were determined in six tolerant and six non-tolerant genotypes. Tolerant plants had the lowest rates of arsenate influx. This was achieved by adaptation of the V_max of arsenate influx with the V_max of the high affinity uptake system saturating at lower substrate concentrations in the tolerant plants. The polymorphism is discussed with relation to adaptation to the extreme environments to which the plants are subjected on mine-spoil soils.     

Copper tolerance in Silene cucubalus

The uptake, translocation and subcellular distribution of copper as well as its effect on chloroplasts and plastocyanin synthesis were studied in a copper-sensitive and a copper-tolerant population of Silene cucubalus (L.) Wib. As a function of time, the copper concentration in roots of tolerant plants increased more slowly than that in roots of sensitive ones. Translocation to the shoot occurred more rapidly in tolerant plants than in sensitive ones. Although it was accumulated in leaf cells, copper was not accumulated in the chloroplasts of either sensitive or tolerant plants. Chlorophyll content was not affected by copper in tolerant plants, whereas sensitive plants became chlorotic. Plastocyanin synthesis was not enhanced as a result of high copper concentrations and no difference in plastocyanin content between tolerant and sensitive plants was detected. Measurements of copper in purified cell walls revealed that storage of the metal in cell-wall material does not play an important role in tolerance mechanism. Uptake characteristics, distribution and cytoplasmic detoxification of copper are discussed.     

Studies on copper and cobalt tolerance in three closely related taxa within the genusSilene L. (Caryophyllaceae) from Zaïre

Summary Experiments were carried out on the tolerance to and uptake of copper and cobalt by three members of a phylogenetic series of taxa within the genusSilene from Zaïre which were reputed to represent a progression of increasing adaptation to metalliferous soils. Plants studied were the widespread and presumably non-tolerantSilene burchelli var.angustifolia (B), a more tolerant variant of this taxon (E) designated an ecotype, and the metallophyteS. cobalticola (C). Studies on the effects of both metals, singly and in combination, on seed germination, seedling and plant performance and yield, and metal uptake from soil cultures, confirmed in general the sequence B<E<C in relation to tolerance of both copper and cobalt. The experimental evidence also supports the ecotypic status of E and points to E and C being relatively more tolerant to copper than to cobalt.     

Can an increased copper requirement in copper-tolerant Mimulus guttatus explain the cost of tolerance? II. Reproductive phase

Circumstantial evidence suggests that plants that have evolved metal tolerance are at a disadvantage on normal soil, i.e. there is a cost of tolerance. One hypothesis for the cause of this cost is that individuals have a greater requirement for copper, and so suffer micronutrient deficiency on normal soils, as a result of a reduced uptake, distribution and/or utilization of copper. We provided highly and less copper-tolerant plants of Mimulus guttatus Fischer ex DC. (the common monkey flower) with sub-optimal copper, and demonstrated the importance of copper as an essential micronutrient during the reproductive phase, both in the production of viable pollen and in seed set. We also looked at the effect of sub-optimal copper supply on the growth of the microgametophyte, and the efficiency with which seed was set. No evidence was found that highly tolerant plants have an increased copper requirement during the reproductive phase. This is in agreement with earlier work on Mimulus guttatus , which investigated the copper requirement of highly tolerant plants during vegetative growth and found that any differences in copper requirement were small. The 'metal requirement hypothesis' is, therefore, not the sole explanation for the cost of copper tolerance in M. guttatus .     

INTER- AND INTRASPECIFIC VARIATION OF MOSSES IN TOLERANCE TO COPPER AND ZINC

Bryophytes are often viewed as slowly evolving with little genetic variation within and among populations. A study of heavy-metal tolerance was initiated to test the capacity of bryophytes to undergo genetic differentiation in response to natural selection. Tolerance of Funaria hygrometrica to copper and zinc was greater in populations that originated on soil with high concentrations of these metals. Protonemal growth was more inhibited by the metals than was germination, and copper was more toxic than zinc. Zinc and copper tolerances were correlated, but so were the zinc and copper concentrations of native substrates. The pattern of population differentiation for heavy-metal tolerance in this species is much like that of flowering plants. Five populations of Physcomitrium pyriforme, which does not occur on metal-contaminated soil, were all highly tolerant of zinc but extremely intolerant of copper. This species seems to have an inherent tolerance to the former. Significant variation in tolerance to copper and zinc occurred among populations, but tolerance did not correlate with metal contents in native substrates. This pattern differs from that of flowering plants. Normal populations of species that colonize contaminated sites tended to be more tolerant than populations of species that do not colonize such sites. The extensive population differentiation in Funaria hygrometrica augments the evidence from electrophoretic data that there is genetic variation among populations of mosses and liverworts.     

Wild plants, <Emphasis Type="Italic">Andropogon virginicus </Emphasis>L. and <Emphasis Type="Italic">Miscanthus sinensis</Emphasis> Anders,are tolerant to multiple stresses including aluminum,heavy metals and oxidative stresses

To isolate high-tolerant plants against aluminum (Al), heavy metals and/or oxidative stresses as a final goal, screening of Al tolerant plants from a collection of 49 wild plants was first of all performed in this study. Andropogon virginicus L. and Miscanthus sinensis Anders showed high Al tolerant phenotypes (more than 35% values in both relative root growth and germination frequency even under 900 μM Al concentration) in our screening. Al tolerance mechanisms in these two plants were characterized and the results suggested that (1) a transport system of toxic Al ions from root to shoot, (2) a suppression of Al accumulation in root tip region and (3) a suppression of oxidative damages by an induction of anti-peroxidation enzymes, such as superoxide dismutase (SOD) and catalase, were involved in the tolerance mechanisms. Six wild plants [Andropogon, Miscanthus, Dianthus japonicus Thunb, Echinochloa crus-galli (L.) Beauv, Reynoutria japonica Houtt, and Sporobolus fertilis (Steud.) W. Clayton] were furthermore tested for their sensitivity against heavy metal stresses and oxidative stresses. The two high Al tolerant plants, Andropogon and/or Miscanthus, showed tolerance to Cr, Zn, diamide or hydrogen peroxide, suggesting common tolerance mechanisms among the tested stresses. Reynoutria showed tolerance to diamide and hydrogen peroxide, Sporobolus to Cr and Echinocholoa to Cd and Cu. Moreover, the collection of wild plants used in this study was a very useful kit to isolate tolerant plants against various abiotic stresses within a short period of time.     

Phytoextraction of cadmium with Thlaspi caerulescens

The in situ phytoextraction of cadmium from soils can only be achieved using plants that are both tolerant to high Cd concentrations and able to extract sufficient amounts of the metal. However, very few plant species are capable of remediating Cd polluted soils in a reasonable time frame. This paper aims to show that the population of the hyperaccumulator Thlaspi caerulescens J. & C. Presl. from Viviez (south of France), which has a high Cd-accumulating capability, is an efficient tool to remove Cd from contaminated soils. Roots of T. caerulescensViviez proliferate in hot spots of metals in soils which is particularly advantageous because of heterogeneity of the distribution of metal in polluted soils. Isotopic techniques showed that plants from this population acquire Cd from the same pools as non-accumulating species, but that it was much more efficient than non-hyperaccumulators at removing the metal from the soil labile pool. This is due: to (i) a specific rooting strategy, and (ii) a high uptake rate resulting from the existence in this population of Cd-specific transport channels or carriers in the root membrane. Growth and overall extraction can be improved with appropriate N fertilisation, supplied either as mineral fertilisers or uncontaminated sewage sludge. Selecting bigger plants is possible from within a suitable Cd-accumulating population to improve the phytoextraction process. Growing the Cd-accumulating populations results in a reduction in the availability of Cd and Zn as shown with field and lysimeter experiments conducted for several years. As a result, on a practical aspect, Cd hyperaccumulating populations of T. caerulescens may be used as a tool to efficiently reduce the availability of Cd in soils, providing appropriate populations are used.     

Accumulation, subcellular localization and ecophysiological responses to copper stress in two Daucus carota L. populations

Copper accumulation, subcellular localization and ecophysiological responses to excess copper were investigated using pot culture experiments with two Daucus carota L. populations, from a copper mine and an uncontaminated field site, respectively. Significant differences of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) concentrations and antioxidant enzyme [superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX)] activities of leaves under Cu treatment were observed between the two populations. At high Cu concentrations (400 and 800 mg kg⁻¹), a significant increase in contents of MDA and H₂O₂ but a significant decrease in activities of SOD, CAT and APX were observed in uncontaminated population. Contrarily, the population from copper mine maintained a lower level of MDA and H₂O₂ but higher activities of SOD, CAT and APX. Copper accumulation in roots and shoots increased significantly with the increase of copper concentrations in soils in the two populations. No significant difference of the total Cu in roots and shoots was found between the two populations at same copper treatment. There were also no striking differences of cell wall-bound Cu and protoplasts Cu of leaves between the two populations. The difference was that Cu concentration in vacuoles of leaves was 1.5-fold higher in contaminated site (CS) population than in uncontaminated site population. Hence, more efficient vacuolar sequestration for Cu and maintaining high activities of SOD, CAT and APX in the CS population played an important role in maintaining high Cu tolerance.